This invention relates to combinations of active substances for use in increasing the potency of a substrate for multidrug resistance related protein.
MRP (multidrug resistance related protein) is a large molecular weight glycoprotein which uses ATP as an energy source and is responsible for the efflux of a specific range of drugs. Other names for MRP are multidrug resistance protein and multidrug resistance associated protein. MRP was discovered because of its over-expression in a number of multidrug resistant human tumour cell lines that do not over-express the MDR1 P-glycoprotein (Cole, S. P. C., et al., (1992); Science (Washington D.C.), 258: 1650-1654). MRP is found in normal human cells, including muscle, kidney and testicular tissue, but levels have been shown to be elevated in many cancer cell lines (Kruh G. D., et al. (1995) Journal of the National Cancer Institute; 87, 16: 1256-1258). The spectrum of substances (including anti-cancer agents) affected by MRP has been broadly defined and includes both non-metabolised anti-cancer agents, such as vincristine (Paul, S., et al., (1996) Proceedings of the National Academy of Science, 93: 6929-6934), and substances possessing a glutathione, glucuronide or sulfate group following cellular metabolism (Jedlitschky, G., et al., (1996) Cancer Research, 56: 988-994). In this way a one or a two-step process may be responsible for the efflux of a particular MRP substrate; an ATP dependent pumping of the agent alone, as is the case with certain anti-cancer agents, including vincristine, or a metabolic conversion followed by the pumping action of MRP, as is seen with glucuronosyl etoposide.
Human cancers have the ability to generate variants resistant to many of the commonly-used chemotherapeutic agents. Combination chemotherapy was introduced to overcome this problem, but variants can arise that are cross-resistant to different sets of drugs; often the drugs within the same set are structurally very different and may act in the cell by totally distinctive mechanisms. Understanding the molecular basis for multidrug resistance (MDR) is an important challenge for cancer research (Clynes, M., (1993) In Vitro Cell. Dev. Biol. 29A: 171-179).
An important goal of experimental chemotherapy research is to identify compounds which, at safe doses, can circumvent drug resistance (Clynes, M. (1993) supra).
Thus, there is a constant search for combinations of drugs for use in chemotherapy so as to circumvent the problem of multidrug resistance. In particular, there is a need for chemotherapeutic agents which can increase the cancer cell killing potency of anti-cancer drugs by inhibiting the various cellular multidrug resistance mechanisms.
The possibility of increasing the activity of etoposide (VP-16) by combining this anti-cancer agent with indomethacin has been investigated by treating murine and human cultured tumour cells with a combination of indomethacin and VP-16 (Maca, R. D. (1991), Anti-Cancer Drug Design 6: 453-466). Maca showed that non-toxic concentrations of indomethacin enhanced the VP-16 sensitivity of a variety of cancer cell lines, and the methotrexate sensitivity of one cancer cell line in vitro. This toxicity enhancement effect with VP-16 was assumed to be due to an increase in the accumulation of VP-16. Non-steroidal anti-inflammatory drugs (NSAIDs) other than indomethacin were not investigated. Furthermore, there is no indication from the results obtained that the effect is seen only with anti-cancer drugs which act as substrates for MRP and with NSAIDs which are of a suitable chemical nature.
The invention provides a combination of an agent which is a substrate for multidrug resistance related protein (MRP) and an inhibitor of MRP which is a non-steroidal anti-inflammatory drug (NSAID) or a structural analogue thereof for simultaneous, sequential or separate use for increasing the potency of said substrate.
Use of a combination of a substrate for MRP and an inhibitor of MRP in accordance with the invention has the potential to overcome the resistance to such substrates exhibited in many conditions, such as resistance to chemotherapy where patients have developed resistance (especially via MRP overexpression to their chemotherapy).
Preferably, the substrate is an anti-cancer drug.
Anti-cancer drugs are also referred to herein as chemotherapeutic drugs.
When the substrate is an anti-cancer drug, the anti-cancer drug is preferably selected from an anthracycline, a vinca alkaloid and an epipodophyllotoxin.
A suitable anthracycline is adriamycin, (also known as doxorubicin), daunorubicin or epirubicin.
A suitable vinca alkaloid is vincristine.
A suitable epipodophyllotoxin is etoposide (VP-16) or teniposide.
Thus, in one embodiment the invention provides a way of increasing the cytotoxic effect of particular anti-cancer drugs by blocking cellular resistance based on MRP.
However, the substrate can also be a transition metal complex, more especially a positive transition metal complex.
When the inhibitor is a structural analogue of an NSAID the structural analogue will normally be derived from an NSAID.
Further, preferably, the NSAID is a heteroarylacetic acid. Suitable heteroarylacetic acids are acemetacin, indomethacin, sulindac, sulindac sulfide, sulindac sulfone, tolmetin and zomepirac.
Alternatively, the NSAID is a non-heteroarylacetic acid compound such as the fenamic acid mefenamic acid.
We have found that the cancer killing effect of several anti-cancer drugs is greatly increased by concurrent administration of non-toxic doses of specific NSAIDs belonging primarily to the heteroarylacetic acid sub-group.
We have found that drugs from other anti-cancer classes, including anti-metabolites, alkylating agents, topoisomerase and microtubule inhibitors do not produce the effects observed with the combinations according to the invention.
As indicated above, preferred inhibitors of MRP are heteroarylacetic acids. These are typically NSAIDs. However, the mechanism whereby heteroarylacetic acids cause their toxicity enhancement effect in accordance with the invention is separate from the mechanism by which NSAIDs are assumed to carry out their therapeutic actions i.e., inhibition of cyclooxygenase enzyme activity. Although not wishing to be bound by any theoretical explanation of the invention, it would appear that the mechanism of toxicity enhancement is dependent on the NSAID fulfilling stringent structural criteria.
The heteroarylacetic acid NSAIDs indomethacin, sulindac and tolmetin as well as the indomethacin analogue acemetacin and the tolmetin analogue zomepirac have been found to enhance the toxicity of MRP-specific substrates. Acemetacin is believed to be a pro-drug of indomethacin and is converted in the liver to the therapeutically active product indomethacin. As we have found that acemetacin is found to be active as a toxicity enhancing agent in vitro in both leukemia and lung cancer cells, where the conversion to indomethacin should not occur, it is clear that the mechanism whereby these compounds cause their toxicity enhancement effect is other than by the mechanism by which NSAIDs are assumed to carry out their therapeutic actions as indicated above.
As evidence that the MRP inhibitor for use in accordance with the invention must meet stringent structural criteria the fact that etodolac, which also belongs to the heteroarylacetic acid group of NSAIDs, does not exhibit the enhanced cytotoxicity observed with other members of this group, as hereinafter described and exemplified.
Mefenamic acid is a fenamic acid NSAID which can be used to enhance the cytotoxicity of MRP substrates in accordance with the invention. However, no additional members of the fenamic acid NSAID chemical class have been shown to enhance toxicity. The fact that close structural analogues of mefenamic acid do not have the ability to enhance MRP substrate toxicity illustrates the extent of the stringency of the structural requirements required for the enhancement of the toxicity of MRP specific substrates.
The following examples demonstrate that the combinations in accordance with the invention have an enhanced toxicity effect. This increased toxicity or cell kill would be expected to kill more tumour cells either totally eliminating cancers (i.e. a cure) or lengthening the time of remission from the effects of the cancer.
It is also expected that the combination in accordance with the invention might broaden the spectrum of cancers treatable with a particular anti-cancer agent.
It is also anticipated that the combination in accordance with the invention would be less sensitive to the evolution of chemotherapeutic drug resistance over time, since fewer cells would survive to produce resistant populations.
Since a given dose of anti-cancer drug would have a greater effect in the combination therapy according to the invention, it would be expected that a reduction in the total dose of chemotherapeutic agent would be possible, thereby reducing the toxic side effects of the treatment.
Resistance to pentavalent arsenicals and trivalent antimonials, as exhibited by Leishmania tropica (a unicellular eukaryotic parasite that causes cutaneous leishmaniasis, oriental sore, in man) is one possible non-cancer disease which might be circumvented by MRP inhibition in accordance with the invention.
However, it is anticipated that other agents, such as antifungal agents, antiparasitic agents, etc. may have their efficacy enhanced when combined with an MRP inhibitor in accordance with the invention.
As regards the use of heteroarylacetic acids as the MRP inhibitor in accordance with the invention, it is known that certain side effects of cancer and the chemotherapeutic drugs used to treat it are caused by prostaglandins. Since all heteroarylacetic acids known to be effective are inhibitors of prostaglandin synthesis, it is reasonable to expect that there would be a reduction in the prostaglandin effects from the chemotherapy.
The substrate for and the inhibitor of MRP for use in accordance with the invention will be administered to a human or animal subject in any form typically used for its administration. Thus, the two components can be administered orally, parenterally or in any other way to achieve the desired effects.